Decoding CRISPR-Cas PAM recognition with UniDesign

Abstract

The critical first step in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (CRISPR-Cas) protein-mediated gene editing is recognizing a preferred protospacer adjacent motif (PAM) on target DNAs by the protein's PAM-interacting amino acids (PIAAs). Thus, accurate computational modeling of PAM recognition is useful in assisting CRISPR-Cas engineering to relax or tighten PAM requirements for subsequent applications. Here, we describe a universal computational protein design framework (UniDesign) for designing protein-nucleic acid interactions. As a proof of concept, we applied UniDesign to decode the PAM-PIAA interactions for eight Cas9 and two Cas12a proteins. We show that, given native PIAAs, the UniDesign-predicted PAMs are largely identical to the natural PAMs of all Cas proteins. In turn, given natural PAMs, the computationally redesigned PIAA residues largely recapitulated the native PIAAs (74% and 86% in terms of identity and similarity, respectively). These results demonstrate that UniDesign faithfully captures the mutual preference between natural PAMs and native PIAAs, suggesting it is a useful tool for engineering CRISPR-Cas and other nucleic acid-interacting proteins. UniDesign is open-sourced at https://github.com/tommyhuangthu/UniDesign.

Keywords: CRISPR–Cas; PAM; UniDesign; computational protein design; gene editing.

Figure 1

The UniDesign workflow for protein–nucleic acid interaction (PNI) design. (A) UniDesign can work for protein–protein interaction design, protein–ligand interaction design, enzyme design and PNI design. (B) The UniDesign pipeline for PNI design comprises four stages (denoted as i, ii, iii and iv).

Figure 2

Visualization of the interactions between PAM nucleotides and PAM-recognizing amino acids. (A) 4UN3 (SpCas9) with TGG PAM; (B) 5F9R (SpCas9) with TGG PAM; (C) 5AXW (SaCas9) with TTGGGT PAM; (D) 5CZZ (SaCas9) with TTGAAT PAM; (E) 6M0W (St1Cas9) with AAAGAA PAM; (F) 5B2O (FnCas9) with TGG PAM; (G) 6JDV (Nme1Cas9) with ATATGATT PAM; (H) 6JE3 (Nme2Cas9) with AGGCCC PAM; (I) 6JOO (CdCas9) with GGGTAAT PAM; (J) 6WBR (AceCas9) with ATACC PAM; (K) 5B43 (AsCas12a) with TTTA PAM and (L) 5XUS (LbCas12a) with TTTA PAM. Nucleotides on nontarget and target strands are shown as yellow and magenta sticks, respectively; nontarget-stranded nucleotides are marked with asterisks. Amino acids are shown in green sticks. Hydrogen bonds are shown as green dashed lines.

Figure 3

UniDesign computed total versus binding energy for all PAM variants on different Cas protein scaffolds. (A) 4UN3 (SpCas9); (B) 5F9R (SpCas9); (C) 5AXW (SaCas9); (D) 5CZZ (SaCas9); (E) 6M0W (St1Cas9); (F) 5B2O (FnCas9); (G) 6JDV (Nme1Cas9); (H) 6JE3 (Nme2Cas9); (I) 6JOO (CdCas9); (J) 6WBR (AceCas9); (K) 5B43 (AsCas12a) and (L) 5XUS (LbCas12a). The variants with consensus PAMs are colored in red while the others are in gray. UEU, UniDesign energy units.

Figure 4

UniDesign predicted consensus PAMs for native Cas proteins. (A) 4UN3 (SpCas9); (B) 5F9R (SpCas9); (C) 5AXW (SaCas9); (D) 5CZZ (SaCas9); (E) 6M0W (St1Cas9); (F) 5B2O (FnCas9); (G) 6JDV (Nme1Cas9); (H) 6JE3 (Nme2Cas9); (I) 6JOO (CdCas9); (J) 6WBR (AceCas9); (K) 5B43 (AsCas12a) and (L) 5XUS (LbCas12a). Note that for Nme1Cas9 and CdCas9, the first two and one positions were excluded from PAM variant generation, respectively, and the ‘-’ symbols were manually added at these positions for plotting sequence logos.

Figure 5

UniDesign predicted PAM-interacting amino acids based on consensus PAM-bearing Cas protein variants. (A) 4UN3 (SpCas9) with four consensus PAM variants; (B) 5F9R (SpCas9) with four consensus PAM variants; (C) 5AXW (SaCas9) with 64 consensus PAM variants; (D) 5CZZ (SaCas9) with 64 consensus PAM variants; (E) 6M0W (St1Cas9) with 32 consensus PAM variants; (F) 5B2O (FnCas9) with four consensus PAM variants; (G) 6JDV (Nme1Cas9) with 16 consensus PAM variants; (H) 6JE3 (Nme2Cas9) with 256 consensus PAM variants; (I) 6JOO (CdCas9) with 432 consensus PAM variants; (J) 6WBR (AceCas9) with 64 consensus PAM variants; (K) 5B43 (AsCas12a) with three consensus PAM variants and (L) 5XUS (LbCas12a) with three consensus PAM variants. Note that 16 and 432 consensus PAM variants were obtained for Nme1Cas9 and CdCas9 because their first two or one PAM positions were not varied, respectively.